The existing technology relates to hepatitis B, an infectious disease caused by hepatitis B virus (HBV) infection, transmitted by blood and body fluid, and characterized by liver damage, which is a serious problem to public health and a great threat to human health. Studies show that some of the patients infected with hepatitis B will develop into a state of chronically persistent infection, which may convert to cirrhosis or primary hepatocellular carcinoma (HCC). China is the high epidemic region of hepatitis B virus infections. 0.35 million people die from hepatitis B associated diseases (such as cirrhosis, HCC etc.) each year, in which the infection rate in the population is 60%, and the carrier rate of the hepatitis B surface antigen (HBsAg) in the population is 10%. Currently, it is estimated that there are about 300 million HBsAg carriers globally, ⅓ of whom living in China. Therefore, the transmission of hepatitis B has become an important issue affecting the population quality in China.
It is shown in practice that hepatitis B vaccination is the best way to control or prevent hepatitis B. The genetically recombinant hepatitis B vaccine has been developed rapidly since 1980s. Since 1981, Merck has successfully developed and commercialized the recombinant vaccine with hepatitis B gene S protein expressed in yeast and used with Alum adjuvant, and has played an important role in preventing and controlling hepatitis B globally. Currently, most marketed hepatitis B vaccines are based on hepatitis B viral S antigen used with Alum adjuvant.
For chronic hepatitis B (CHB) patients, persistent HBV replications in hepatocyte will cause the exhaustion of the virus-specific T cell in the body and the immune escape of the virus, which will lead to immune tolerance of the patients, and weaken the function of virus-specific CTL. Studies have demonstrated that the low response of HBV specific T cell may be one of the most important reasons for the persistent infection of HBV, the specific molecular mechanism of which is still unknown. It is anticipated that it might be associated with load of virus antigen, efficiency of innate immunity, type of antigen present cells, quantity and function of T helper cell and regulatory T cells, and regulation of costimulatory molecules.
According to the Guideline for Prevention and Treatment of Chronic Hepatitis published by Chinese Medical Association of liver diseases and infectious diseases, the HBV patients who are suitable for anti-virus agent treatments should be provided with antiviral treatment. Antiviral agents against hepatitis B currently include alpha interferon (α-IFN) and nucleos(t)ide analogues such as lamivudine, adefovir, entecavir, telbivudine, tenofovir etc., which can inhibit the copy number of HBV DNA in patients within the scope of indication, but are easy to develop drug resistance upon long-term administration. Moreover, the discontinuation of nucleos(t)ide analogues administration will lead to reoccurrence even exacerbation of the disease. Long-term administration of interferon will lead to significant side effects due to its bone marrow suppression effects. Formulations of Glycyrrhizin, Silymarin, polyunsaturated lecithin and bicyclol all have effects on anti-inflammation, anti-oxidation, protection of hepatocyte membrane and organelles in different levels, and it is shown by clinical trial results that they can improve the biochemical index of liver, but cannot replace anti-virus therapies.
Currently researchers believe that the effective immunotherapy should rely on stimulating immune system of hepatitis B carriers. It is known that Granulocyte-macrophage colony stimulating factor (GM-CSF) is a type of important growth factor of hematopoietic cells with multiple potentials, and has a significant curative effect on leukopenia caused by various reasons. GM-CSF, mainly produced by activated T cells, B cells, macrophage, mast cells, endothelial cells and fibroblasts, can not only promote proliferation, differentiation and maturation of hematopoietic precursors, but also have different levels of stimulating effects on other cells such as antigen presenting cell (APC), fibroblasts, keratinocytes, skin mucous cells. etc. In 1993, Dranoff et al. used GM-CSF as an immune adjuvant to enhance immune response of cancer vaccine for the first time. The enhanced immune effects of GM-CSF may rely on the enhanced ability of antigen presentation by APC. When interacting with Dendritic Cells (DC), GM-CSF can promote antigen presentation [1], increase IL-2 production, activate CD4+ T cells, increase the ability of antibody secreting and enhance the function of CD8+ T cells [2]. Recent investigations indicated that GM-CSF can activate T cells and endothelia, enhance the function of APC, upregulate molecular MHC, costimulate molecules, participate immune modulation of organism, and enhance the therapeutic effects of antiviral agents. However, Hasan et al. discovered that GM-CSF did not provide significant adjuvant activity, i.e. it could not effectively enhance primary immune response, when it was intramuscularly injected immediately before the injection of recombinant hepatitis B vaccine in normal individual [3]. V. Bronte found that systemic high level of GM-CSF can induce transient T cells suppression [4]. In a phase II clinical trial for prostate cancer vaccine, S. J. Simmons et al. used GM-CSF as systemic adjuvant, but could not detect the enhanced clinical responses after the injection of DC-polypeptide or significantly enhanced immune responses. Moreover, the dose related side effects such as local reactions, fatigue, bone pain, myalgia and fever occurred in some patients [5]. Such results are different from some other reports in which GM-SCF acting as immune adjuvant could significant enhance antigen specific immune responses, which indicates that the doses, administration duration, and immune dose of GM-CSF are closely related to clinical immune results. However, the studies on dose and duration of GM-CSF immunotherapy are not thorough, and it is necessary to perform systematic studies to optimize the administration protocol of GM-CSF as immumotherapy agent.
It has been reported that using interferon alone as treatment for HBV could achieve about 25%-40% of efficacy. Lamivudine is still the primary choice as treatment for HBV infection in most regions due to its relative safety and low price, although the rapidly developed drug resistance is the main drawback [6]. As the usage of adefovir increases, the drug resistance to such agent has become a major problem, which indicates that some patients do not respond to the mono-agent therapy, or are easy to develop drug resistance. Given the successful combinational therapy against HIV infections and the various problems associated with mono-agent therapy against HBV, more researchers began to study combinational therapy against HBV [7]. When conducting combination therapy with GM-CSF and interferon, Guptan et al. observed that 60% of the HBV patients, who did not respond to the interferon monotherapy, had a decreased level of HBeAg and HBV-DNA at the end of the initial combination therapy, but some of the patients showed recurrences of the virus [8]. While after six months' combination therapy with interferon and hepatitis B vaccine, Heintges et al. observed that 50% (8/19) of the individuals, who did not respond to interferon monotherapy, showed undetectable HBV-DNA level, but the sustained response rate after the therapy was not reported in the clinical trial. Some studies reported that by direct treatment with HBsAg vaccination, 28.6% of the virus carriers had reduced level of virus replications and 21.4% of the virus carriers had negative HBV-DNA. However, Dikici et al. found that there was no significant difference between the hepatitis B vaccinated group and the unvaccinated group of the chronic HBV infected children with immune resistance.
The Chinese publication CN 1990043A “Application of recombinant human granulocyte macrophage colony stimulating factor in the treatment or prevention of hepatitis B virus” disclosed the combination administration of recombinant human GM-CSF and genetically engineered Hepatitis B vaccine can enhance humoral immune response of organism. The administration of recombinant human GM-CSF before genetically engineered HBsAg vaccination can stimulate the cellular immunity in animals, promote T cell differentiation, stimulate the secretion of cellular factors such as IFN-γ and the like in Th1 cell, increase the production of IgG2a antibody, and enhance the function of cytotoxic T cells (CTL), so that a treating efficacy for HBV is achieved. In recent years, different kinds of cytokines and chemokines have been used as the immune adjuvant for the studies of animal models and human vaccines to promote antigen recognition and T cell proliferation. It is also reported that GM-CSF is currently the most used cytokine adjuvant in terms of increasing the immunogenicity of cancer vaccines. GM-CSF can release the cytokine by genetic transducing into tumor cells or to surrounding normal cells. In addition, GM-CSF can be used locally or systemically for different vaccinations on animals or patients administered with the form of recombinant protein. However, it is still under argument whether GM-CSF should be used as an immune adjuvant for anti-virus vaccine in human. By intramuscularly injecting GM-CSF immediately before the injection of recombined hepatitis B vaccine into normal individual, Hasan et al. found that GM-CSF cannot provide significant adjuvant activities, which indicates it cannot enhance primary immune responses effectively [3]. The difference in results by using the same GM-CSF as adjuvant may relate to the dose, injection site and method of immunization in actual application.
Based on the studies listed above, the inventors of the present application propose to provide a new pharmaceutical composition for viral immunotherapy, especially a pharmaceutical composition for viral immunotherapy for persistent hepatitis B infection.
The existing techniques associated with the present disclosure are:
1. van de Laar L, Coffer P, Woltman A: Regulation of dendritic cell development by GM-CSF: molecular control and implications for immune homeostasis and therapy. Blood 2012, 119(15):3383-3393.
2. Wanjalla C, Goldstein E, Wirblich C, Schnell M: A role for granulocyte-macrophage colony-stimulating factor in the regulation of CD8(+) T cell responses to rabies virus. Virology 2012, 426(2):120-133.
3. Cruciani M, Mengoli C, Serpelloni Mazzi R, Bosco O, Malena M: Granulocyte macrophage colony-stimulating factor as an adjuvant for hepatitis B vaccination: a meta-analysis. Vaccine 2007, 25(4):709-718.
4. Paola Rizza, Maria Ferrantini, Imerio Capone, Filippo Belardelli: Cytokines as natural adjuvants for vaccines: where are we now. Trends in Immunology, 2002, Vol. 23, No. 8, 381-383.
5. S. J. Simmons, B. A. Tjoa, M. Rogers, A. Elgamal, G M. Kenny, H. Ragde, M. J. Troychak, A. L. Boynton, G P. Murphy, GM-CSF as a Systemic Adjuvant in a Phase II Prostate Cancer Vaccine Trial. The Prostate, 1999, 39:291-297.
6. Morrey J, Bailey K, Korba B, Sidwell R: Utilization of transgenic mice replicating high levels of hepatitis B virus for antiviral evaluation of lamivudine. Antiviral research 1999, 42(2):97-108.
7. Paul N, Han S-H: Combination Therapy for Chronic Hepatitis B: Current Indications. Current hepatitis reports 2011, 10(2):98-105.
8. Rajkumar C G, Varsha T, Seyed N K, Shiv K S: Efficacy of granulocyte-macrophage colony-stimulating factor or lamivudine combination with recombinant interferon in non-responders to interferon in hepatitis B virus-related chronic liver disease patients. Journal of Gastroenterology and Hepatology 2002, 17.